Small interfering RNA (siRNA) are a promising class of drugs that are limited by delivery challenges. Naked siRNA has poor cellular uptake and rapid degradation in vivo, thus siRNA is typically administered in nanoparticle (NP) formulations. To circumvent extracellular barriers of degradation and poor uptake as well as intracellular lysosomal trafficking, our lab pioneered the development of pH-responsive cationic diblock tercopolymer nanoparticles (pHCNPs) for the delivery of siRNA, which greatly improve siRNA therapeutic efficacy. Local in vivo delivery of siRNA-pHCNPs has shown promise in bone and other tissues, but many diseases, including many of the musculoskeletal system require systemic administration. Systemic delivery of NPs is hampered due to serum adsorption, leading to mononuclear phagocyte system (MPS) uptake and poor circulation times, and may potentiate immunogenicity. In fact, many siRNA-NP therapeutics that are approved or in late stages of development take advantage of MPS accumulation in liver, targeting hepatic diseases. Reducing protein-NP interactions is key to improving the systemic delivery of siRNA-NPs to reach other target tissues. Poly(ethylene glycol) (PEG) modification (PEGylation) of NPs is the current standard to reduce protein adsorption, but it also reduces NP efficacy by hampering uptake and may induce immunological responses due to anti-PEG antibodies. Zwitterionic (ZI) moieties have shown great promise in reducing protein adsorption and are less disruptive to NP functional characteristics than PEG. Biomimetic ZI peptides (ZIPs) are a promising new approach to improve polymeric NP pharmacokinetic properties. In particular, semi-randomized ZIPs (srZIPs) allow testing of charge sequence semi-independently of amino acid composition. These attributes altogether lead to the hypothesis that semi-randomized ZIPs (srZIPs) designed with low aggregation potential can be used to modify NPs to improve systemic circulation and siRNA delivery by reducing NP-serum protein interactions. We will test this hypothesis in three ways. In Aim 1, we will generate a library of srZIP-pHCNP conjugates to test the effects on serum-induced aggregation compared to nave pHCNPs and PEG-pHCNPs. In Aim 2A, we will test these conjugates in vitro using target cells (mesenchymal stem cells) and in MPS cells (macrophages), and in Aim 2B we will evaluate improvements in circulation times of the pHCNP conjugates in vivo. In Aim 3 we will use an established mouse femur fracture model to investigate fracture accumulation of pHCNP bearing therapeutic anti-WWP1 siRNA (WW Domain Containing E3 Ubiquitin Protein Ligase 1, a negative regulator of fracture healing), which we have shown to expedite bone fracture healing in using a local delivery approach. At the completion of this project, we expect to identify new peptide-based anti-aggregation approaches for the delivery of siRNA in polymeric nanoparticles, laying the foundation for future systemic delivery of siRNA for musculoskeletal applications and beyond.